This invention relates to the field of detecting a genetic marker of T-cell malignancies. The invention provides compositions and methods useful for detecting clonal T-cell receptor gene arrangements. By using PCR and temporal temperature gradient gel electrophoresis (TTGE), the clonality of T-cell populations in a sample can be determined.
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
T lymphocytes are important players in the immune response of mammalian organisms. Each T-lymphocyte has on its surface a molecule, known as the T-cell receptor (xe2x80x9cTCRxe2x80x9d) that exhibits a specificity of binding to a target antigen that is similar to the specificity of binding exhibited by antibodies, and TCR is encoded by genes having an organization similar to that of antibody genes.
During normal T-cell development, each TCR gene (xcex1, xcex2, xcex3 and xcex4) can rearrange, leading to highly diverse TCR proteins. Among the four TCR gene types, the TCR-xcex3 genes are the first to rearrange. During the rearrangement of each TCR gene, one of its different variable (V) regions combines with one of its different joining (J) regions. In the TCR-xcex3 gene, a few nucleotides (N region) are also inserted randomly into the VJ regions by terminal nucleotidyl tansferase (TdT), resulting in an increased sequence diversity in the TCR-xcex3 gene as compared to the other TCR genes. It is believed that each mature T-cell possesses an individual sequence of rearranged TCR genes.
T-cell malignancies can present to the physician with strikingly similar clinical patterns to simple reactive, or inflanmatory, diseases. T-cell malignancies, however, can be differentiated from reactive diseases by the presence in malignancy of an overgrowth of a single, clonal, T-cell population. Therefore, analysis of TCR gene rearrangement, and particularly the TCR-xcex3 gene, is of practical value in determining the clonality T-cell populations for the diagnosis and prognosis of T-cell malignancies (Flug et al., Proc Nat1 Acad Sci USA. 82: 3460-3464, 1985; Yanagi et al., Nature 308: 145-149, 1984; Waldmann et al., N Engl J Med 313: 776-783, 1985; Raulet, Annu Rev Immunol. 7: 175-207, 1989; Theodorou et al., Blood 86: 305-310,1995).
To date, numerous techniques have been used to analyze TCR gene rearrangement. Southern blotting hybridization was the first widely used technique (Spagnolo et al., Pathol. 26: 268-275, 1994). Because Southern blotting hybridization can be time-consuming and labor-intensive, and requires the use of large amounts of high molecular weight DNA and radioactivity, polymerase chain reaction (PCR)-based techniques have become more popular in research and clinical laboratories. These techniques can also permit the use of DNA extracted from formalin fixed parafin-embedded specimens and are more rapid than hybridization approaches (Theodorou et al., Blood 86: 305-310,1995; Murphy et al., J Cutan Pathol. 27: 228-234, 2000; Anderson et al, J. Cutan Pathol. 26: 176-182, 1999; Wood et a., J Invest Dermatol. 103: 34-41,1994; Menke etal., Electrophoresis 16: 733-738, 1995; Bourguin et al., Proc Natl Acad Sci USA. 87: 8536-8540, 1990).
PCR amplification of one or more TCR genes from non-malignant peripheral blood or lymph node tissue samples generates a mixture of multiple DNA molecules differing in size and/or base pair composition. Because the combinatorial and junctional diversity in TCR gene rearrangement is limited, the amplified products may be of similar length. Thus, the limited diversity may result in false clonal bands when analyzed by a standard gel electrophoresis methods that separate DNA molecules based solely on size (Theodorou et al., Blood 86: 305-310,1995; Menke et al., Electrophoresis 16: 733-738, 1995), and can occur when using either Southern blotting hybridization (which is based on agarose gel electrophoresis) or PCR followed by standard polyacrylamide gel electrophoresis (PAGE). Furthermore, due to the poor resolution of standard gel electrophoresis methods, DNA bands of low frequency clones may be lost in the polyclonal background xe2x80x9csmear,xe2x80x9d leading to difficult in interpreting the results of such an analysis.
Recently, electrophoresis techniques that resolve DNA molecules based on size and base pair composition have been explored, such as polymorphism (SSCP) (Murphy et al., J Cutan Pathol. 27: 228-234, 2000), denaturing gradient gel electrophoresis (DGGE) (Theodorou et al., Blood 86: 305-310, 1995; Anderson et al., J. Cutan Pathol. 26: 176-182, 1999; Wood et al., J Invest Dermnatol. 103: 34-41, 1994) and temperature gradient gel electrophoresis (TGGE) (Menke et al., Electrophoresis 16: 733-738, 1995). To maximize the resolution of SSCP, however, more than one electrophoretic condition is often needed (Orita et al., Genomics 5: 874-879, 1989). Moreover, although DGGE has been gaining popularity to determine the clonality of T-cell populations (Theodorou et al., Blood 86: 305-310,1995; Anderson et al., J. Cutan Pathol. 26: 176-182, 1999; Wood et al., J Invest Dermatol. 103:34-41, 1994), the difficulty in preparing denaturing gradient polyacrylamide gel limits the routine usage of this technique in clinical laboratories. Similarly, the routine use of TGGE in clinical laboratories is also limited because of its reliance on an instrument that is both expensive and difficult to maintain. (Menke et al. Electrophoresis. 16:733-738, 1995; Alkan et al. Arch Pathol Lab Med. 125:202-207, 2001).
Thus, there remains in the art a need for methods and compositions that can reproducibly and economically resolve clonal T-cell receptor populations in patient samples.
In the present invention provides methods and compositions to determine the clonality of T-cell receptor populations present in a sample. Temporal temperature gradient gel electrophoresis (TTGE), developed by Yoshino et al. (Nucleic Acids Res. 19:3153, 1991) and Chen et al. (Clin Chem. 45:1162-1167, 1999), can be used to detect the presence or absence of a clonal TCR gene rearrangement. In the instant methods, target DNA is electrophorcsed in a denaturing gel, and the temperature of the gel is increased gradually and uniformly across a range in which differences in the base composition of the target DNA are resolved. Because the gel itself is not a gradient gel, and because the temperature gradient is temporal rather than spatial across the gel, the instant methods can be performed in an economical fashion. In addition, the resulting DNA band patterns can be easily interpreted and quality controlled in a clinical laboratory setting.
In accordance with the present invention, TTGE can detect clonal TCR (e.g., TCR-xcex3) gene arrangements present in a sample at concentrations as low as one malignant T-cell among 100 normal cells. Additionally, DNA can be extracted from stored samples (e.g., samples stored at 4xc2x0 C. for up to 7 days and at room temperature for up to 4 days) and used for PCR amplification to generate gene amplicons for TCR gene rearrangement analysis by TTGE without significant variability of the band pattern and signal intensity. Thus, the invention provides simple, accurate and sensitive techniques to diagnose and monitor patients with T-cell malignancies.
In a first aspect, the instant invention relates to methods for determining the clonality of a T-cell receptor (TCR) rearrangement in a sample comprising the steps of extracting nucleic acids from the sample; amplifying the nucleic acids, preferably by polymerase chain reaction with one or more TCR specific primers to provide one or more TCR DNA fragments; and analyzing amplified TCR DNA fragments using an electrophoretic gel by temporal temperature gradient gel electrophoreis (TTGE). The present of one or more discrete bands in the electrophoretic gel indicates the presence of a clonal TCR rearrangement.
In certain embodiments, the electrophoretic profile of a test sample can be compared to one or more control samples (e.g., a negative control sample obtained from a sample not containing a clonal T-cell rearrangement), and the presence of one or more discrete bands in the test sample that are not present in the control sample(s) indicates the presence of a clonal TCR rearrangement.
As used herein, the term xe2x80x9cT-cell receptorxe2x80x9d refers to the antigen-recognition molecules present on the surface of T-cells. See, e.g., Garcia et al., Rev. Immunogenet. 1999;1(1):75-90. As indicated above, during normal T-cell develop, each of the four TCR genes, xcex1, xcex2, xcex3 and xcex4, can rearrange leading to highly diverse TCR proteins. See, e.g., Gill and Gulley, Hematol. Onco Clin. North Am. 1994 August;8(4):751-70; Greiner, Am. J. Pathol. 1999 January;154(1):7-9.
The term xe2x80x9cclonalityxe2x80x9d as used herein regarding T-cells refers to the expansion of a single population of T-cells in a sample. Such a clonal expansion occurs when, for example, T-cell populations are challenged by an antigen. In response, that population of T-cells that recognize the antigen increase in number. In more dramatic fashion, clonal expansion occurs in certain T-cell malignancies, in which a single T-cell population expands beyond normal levels due to a loss of growth control in a T-cell. In certain embodiments, clonality is determined by analyzing a TCR-xcex1, TCR-xcex2, TCR-xcex3 and/or TCR-xcex4 gene rearrangement using PCR amplification with two or more appropriate primer sequences for the gene(s) of interest, and temporal temperature gradient gel electrophoresis, as described herein
The term xe2x80x9camplifyxe2x80x9d with respect to nucleic acid sequences refers to methods that increase the representation of a population of nucleic acid sequences in a sample. Nucleic acid amplification methods, such as PCR, isothermal methods, rolling circle methods, etc., are well known to the skilled artisan. See, e.g., Saiki, xe2x80x9cAmplification of Genomic DNAxe2x80x9d in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Cailf. 1990, pp 13-20; Wharam et al., Nucleic Acids Res. Jun. 1, 2001;29(11):E54xe2x80x94E54; Hafner et al., Biotechniques 2001 April;30(4):852-6, 858, 860 passim; Zhong et al., Biotechniques 2001 April;30(4):852-6, 858, 860 passim.
In preferred embodiments, generic DNA representing a TCR gene is extracted from a cell and amplified; however, other nucleic acids (e.g., mRNA) may also be extracted and amplified to perform the clonality assays of the present invention.
In certain embodiments, nucleic acids can be amplified using PCR with TCR-specific primers, i.e., oligonucleotides suitable for amplifying specific regions of TCR Preferred primers are Vxcex31xe2x88x928 (5xe2x80x2-AGGGTTGTGTTGGAATCAGG-3xe2x80x2) (SEQ ID NO:3) for V region and Jxcex3xc2xd (5xe2x80x2-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCCTGTTCCACTGCCAAA GAGTTTCTT-3xe2x80x2) (SEQ ID NO:4) for J region. The primer sequence for Jxcex3xc2xd for J region is a GC-clamp region designed to introduce a high melting point domain at one end of the PCR amplicons, facilitating analysis by TTGE. Additional suitable primers are described below in the figures.
As used herein, the term temporal temperature gradient gel electrophoresis (xe2x80x9cTTGExe2x80x9d) refers to electrophoresis methods in which samples are run in a gel matrix, e.g., a polyacrylamide gel, and the temperature of the gel is altered in a time-dependent fashion as the samples are migrating through the gel. In such a gel, the relative mobility of a DNA sample is based on its relative thermal stability. Thus, by careful selection of the temperature profile of the gel, the migration of molecules in the sample can be altered as the temperature changes. For example, DNA molecules can be run in the gel matrix using a temperature profile in which certain base pairs in the molecules xe2x80x9cmelt;xe2x80x9d that is, where the temperature is sufficient to disrupt the hydrogen bonding in the Watson-Crick base pairs. The skilled artisan will understand that different DNA sequences will have different relative thermal stability; e.g., A-T-rich regions will melt at a lower temperature than G_C-rich regions. As the base pairs melt, the molecular structure of the DNA becomes locally xe2x80x9copen,xe2x80x9d resulting in a change in mobility.
The term xe2x80x9cgelxe2x80x9d as used herein refers to any semisolid medium capable of supporting a fluid phase for separating nucleic acids based on size and melting temperature profile of the individual nucleic acid molecules when an electric field is placed across the medium. Gels may include starch, acrylamide, agarose, or mixtures of these materials, and may be a single concentration, or gradient. The electrolyte used to separate materials in a gel may be either continuous or discontinuous. In preferred embodiments, the gel is an acrylamide gel of a single concentration throughout, and the elecrolyte is a continuous buffer system.
Typical etectrophoresis gels comprise a plurality of channels, known to the artisan as xe2x80x9clanes,xe2x80x9d in which individual samples (and/or controls) may be loaded and run in parallel on the same gel. Because all such samples are exposed to essentially the same electrophoresis conditions, the characteristics of samples (and/or controls) in different lanes can be directly compared. Molecules that migrate to the same location in the gel appear, upon detection, as xe2x80x9cbands;xe2x80x9d i.e., a line of material about the width of the lane and having a characteristic height (often about 0.2 to about 5 mm). Bands on separate gels can often be compared by determining the relative location of the band in relation to controls (xe2x80x9cstandardsxe2x80x9d) that have been run in each of the separate gels.
Material will migrate to the same location in the gel for a number of reasons, depending on the type of electrophoresis used. For example, in isoelectric focusing, material will migrate to the same location if the material has the same isoelectric point. As discussed herein, in TTGE, DNA molecules will migrate at the same rate, and thus migrate to the same location in a gel, if the molecules have the same relative thermal stability.
As used herein, the term xe2x80x9cthe presence one or more discrete bandsxe2x80x9d refers to a signal that is obtained from molecules that migrate to a discrete location in an electrophoretic gel. For example, a clonal T-cell rearrangement in a malignancy can be detected as a population of DNA molecules that migrate at identical rates through a gel, such that they appear as a single xe2x80x9cbandxe2x80x9d upon detection, e.g., by gel staining techniques well known in the art. As discussed herein, because genes are xe2x80x9cmultiallelic;xe2x80x9d that is, there are two alleles of each gene, one on each chromosome of a chromosome pair, a clonal T-cell rearrangement may appear as more than one discrete band in such a gel.
Preferably, a discrete band provides a signal that is twice the background signal 3 times the background signal, 4 times the background signal, 5 times the background signal, 7.5 times the background signal, 10 times the background signal, 15 times the background signal, 20 times the background signal, 50 times the background signal, 100 times the background signal, and 500 times the background signal, where the background signal is obtained from a negative control sample or from an area of the electrophoresis lane containing diffuse staining.
Additionally, a discrete band can be discriminated from a diffuse xe2x80x9csmearxe2x80x9d within a gel by a sharp fall off in intensity present at the band boundary, where signal intensity rapidly falls to background. This rapid fall-off results in a band that is between {fraction (1/10)}th and twice the height of a band obtained from a positive standard run in the same gel, while diffuse staining results in a band that is greater than twice the height of the positive standard.
The term xe2x80x9csamplexe2x80x9d as used herein refers to any liquid or solid material believed to comprise T-cells. In preferred embodiments, a sample is a tissue sample from an animal, most preferably a human. Preferred sample tissues of the instant invention include, but are not limited to, plasma, serum, whole blood, blood cells, lymphatic fluid, lymphatic cells, lymph node tissue, cerebrospinal fluid, and skin or other organs (e.g., biopsy material). Such sample tissues may or may not be fixed, e.g., by formalin, and/or embedded for sectioning, e.g., in paraffin.
The term xe2x80x9cpatient samplexe2x80x9d as used herein refers to a sample from an animal, most preferably a human, seeking diagnosis or treatment of a disease.
In another aspect, the present invention relates to methods for diagnosing a patient suspected of having a neoplastic T-cell disease. In these methods, a clonal expansion of a T-cell population is determined by analyzing the clonality of TCR gene rearrangements in a sample. A positive indication of such a clonal expansion is indicative of a neoplastic T-cell disease. These methods preferably comprise obtaining a sample from a patient and determining whether a clonal TCR gene rearrangement is present in the sample using TTGE, wherein the presence of a discrete band in the clectrophoretic gel indicates the presence of a clonal TCR rearrangement.
In certain embodiments, these methods can be used to compare two or more lesions in a patient having a neoplastic T-cell disease. As used herein, the term xe2x80x9clesionxe2x80x9d refers to diseased tissue in a patient. In these embodiments, a sample from a first lesion and a second lesion in the patient can be obtained, and each sample can be analyzed for the presence or absence of identical clonal TCR gene rearrangements in each sample. In various preferred embodiments, the lesions can be obtained from the same disease focus, but at different times during the course of a disease (e.g., before and after a treatment regimen); or from different disease foci at the same or different times. As used herein, the term xe2x80x9cdisease focusxe2x80x9d refers to a spatially unique location in a patient that harbors diseased tissue.
In yet another aspect, the present invention relates to methods and compositions for designing a treatment regimen. In these methods, the presence or amount of a clonal TCR gene rearrangement in a patient following a selected treatment(s) can be used to assess the success or lack thereof in the treatment regimen. Such methods can also compare the relative presence or amount of a clonal TCR gene rearrangement in a patient before and after such a treatment regimen.
Similarly, the present invention also relates to methods and compositions for screening therapeutic compounds. In these methods, the presence or amount of a clonal TCR gene rearrangement in a patient following administration of one or more compounds can be used to assess therapeutic efficacy. Such methods can also compare the relative presence or amount of a clonal TCR gene rearrangement in a patient before and after administration of one or more compounds.
In accordance with still another aspect, the present invention relates to methods for preparing a substantially pure nucleic acid migration marker for use in a TTGE gel, and to the nucleic acid migration markers themselves. Preferably, such a marker can be prepared from one or more DNA fragments having known migration rates in the TTGE gel. For example, a standard DNA ladder for TTGE analysis can be made by amplifying one or more DNA fragments containing known TCR rearrangements using TCR-specific primers. Alternatively, synthetic methods well known to the skilled artisan can be used to prepare such a marker.
In either case, the DNA fragments can then be inserted into a plasmid cloning vector. The plasmid can then be used to transform a cell population in order to amplify the number of plasmid molecules, thus preparing xe2x80x9cplasmid amplicons.xe2x80x9d Isolated plasmids and/or amplicons can be used to generate a large number of DNA migration markers, e.g., by PCR using TCR-specific primers.
In certain embodiments, the plasmids and/or amplicons containing known TCR rearrangements can be subjected to PCR in parallel with samples to be analyzed for clonal TCR rearrangement, using identical conditions and TCR-specific primers. In these embodiments, the plasmid amplicons can both provide both DNA migration markers and a positive PCR control, in which the presence of the DNA migration marker in the TTGE gel confirms that the PCR reaction successfully occurred in the samples.
Mixtures of a plurality of TTGE migration markers can provide multiple reference points in a TTGE gel. Thus, in preferred embodiments, a composition of migration markers comprises 2, 3, 4, 5, 6, 7, 10, and 20 different migration markers in a buffer suitable for loading on a TTGE gel.
The term xe2x80x9cmigration markerxe2x80x9d as used herein refers to a nucleic acid sequence known to migrate with a certain relative mobility in a TTGE gel and form a discrete band in the gel. The term xe2x80x9csubstantially purexe2x80x9d as used herein regarding TTGE migration markers refers to a purity sufficient to provide such a discrete band on a TTGE gel.